The fruit fly Drosophila melanogaster h a s b e e n a leading model s y st e m to stud y genetics a n d development a l biology. The large m ut a t i o n a l s c reens per fo r med in t h e 1980s (Lewis et al. 1980;Nusslein-Volhard a n d Wieschaus 1980;Spencer et al. 1982; Sc hupba c h a n d Wieschaus 1986; St Johnston 2002), together with t h e pletho ra of effective genetic tools (Duffy 2002;del Valle Rodriguez et al. 2011), revealed t h e functions of m a n y genes, gene regulatory networks, a s well a s d e m o n s t r a t e d how organ isms t h a t a r e phenotypically u n r e l a t e d s h a r e a large proportion of the ir g enes a n d f u n d a m e n t a l molecular a n d cellular fun ctions (Holley et al. 1995;Pearse a n d Tabin 1998). The introduction of t h e CRISPR/Cas9 s y s t e m for tar g e t e d a n d precise g e n o me editing of D. melanogaster (Gratz et al. 2013) provided a n efficient new syst e m to directly m a n i p u l a t e genes a n d s t u d y the ir influence on or-N a k a m u r a et al. 2007;Kagesawa et al. 2008;Schaeffer et al. 2008;Markow et al. 2009;Niepielko et al. 2011;Werner et al. 2018Werner et al. , 2020)).
Studying t h e evolution of species a t t h e molecular level is restricted by t h e availability of their high-quality gen om e assemblies.
A few s eq uen ce d Drosophila species h av e limited tools for genetic ana lyses (i.e. Holtzman et al. 2010;Werner et al. 2010;del Valle Rodriguez et al. 2011; Niepielko a n d Yakoby 2014; Stern et al. 2017), which p re se n t s a n i m pe d i m en t to u nd er st a n d i ng t h e evolutionary m e c h a n i s m s responsible for c om mo n a n d u n i q u e organismal traits. The im pr ov em en t of sequencing technologies, including long-read sequen cing via Pacific Biosciences (PacBio) a n d Oxford Nanopore, h av e allowed for massive efforts to s eq u en ce t h e geno mes of m a n y different species, including Drosophila (Kim et al. 2020), a s well a s upda tin g a n d improving t h e contiguity a n d compl eteness of existing ass emblies (Paris et al. 2020). These advan ces gan i smal pheno t yp e s without t h e tedious m u t a t i o n a n d a re m o n u m e n t a l in furthering t h e development of biological sysscreening cycles. At t h e s a m e time, t h e r e a r e t h o u s a n d s of other Drosophila species with interesting differences in behavior, chromo s o m a l a r r a n g e m e n t , gene expression, pigmenta tion, diverse t e m s in other Drosophila species, which a r e t h e stepping stone to stu dy m e c h a n i s m s of evolutionary diversity.
Since t h e discovery of a white-eyed fruit fly in 1910 by T h o m a s cell signaling, a n d fascinating morphologies (Spieth 1952; H u n t Morgan (Morgan 1910), t h e w gene h a s b e e n extensively Significance s t a t e m e n t Morphological a n d p a t t e r n i n g diversities a r e c o m m o n in n a t u r e . However, t h e m e c h a n i s m s underlying t h e s e evolutionary differences h a v e b e e n s t u d i e d only in a limited n u m b e r of an i m a ls . High-throughp ut tools h a v e b e e n created to s t u d y de v el opm e n t a n d evolution, yet t h e a b s e n c e of high-quality g e n o m e s e q u e n c e s for m a n y org anisms of i n t e r e s t h a s b e e n a n obstacle to t h e exploration of m e c h a n i s m s controlling diversity in n a t u r e . Here, we g e n e r a t e d t h e first high-quality g e n o m e s e q u e n c e of Drosophila nebulosa a n d employed g e n o m e engineeri ng to t e s t w h e t h e r vision is necessa ry for matin g . s t u d i e d in D. melanogaster. The gene enco des a n ATP-binding cassette transp o rter, which forms h e t e r o d i m e r s with t h e Scarlet or Brown proteins to deliver p i g m e n t precur sors into p i g m e n t cells, consequ ently making r e d eyes in wild-type flies (Sullivan a n d Sullivan 1975; Sullivan et al. 1979). Mutation of th is gene m a y result in t h e alteration of t h e protein s t r u c t u r e a n d lead to loss of function, resulting in white-eyed flies (Mackenzie et al. 1999). Due to t h e simplicity of identification, eye-color of w-disrupted flies is frequently u s e d a s a selectable m a r k e r for transgenic flies. However, deleter ious effects d u e to t h e loss of w h a v e b e e n uncovered in t h e p a s t decades. Several st ud i e s h a v e d o c u m e n t e d alteration s in courting, copulation success, exploratory behavior, visual acuity, learning a n d m e m o r y of t h e r m a l stress, a n d sexual preference in D. melanogaster overexpressing or deficient of t h e White protein (Anaka et al. 2008; S i t a r a m a n et al. 2008; Krstic et al. 2013;Ferreiro et al. 2017;Xiao et al. 2017). Behavioral changes, s u c h a s t h e s e h a v e b e e n shown to b e t h e resu lt of a l tered levels of specific n e u r o t r a n s m i t t e r s , s u c h a s serotonin a n d d o p a m i n e (Becnel et al. 2011;Ries et al. 2017), whose precursors a r e t r a n s m i t t e d by t h e White protein (Krstic et al. 2013;Xiao et al. 2017). Other s t u d i e s h a v e rep o rted t h a t w -disrupted D. melanogaster lack optical insulation provided by eye p i gment a n d t h u s show i m p a i r e d visual acuity (Ka lmus 1943), in creased light sensitivity (Wu a n d Wong 1977), deficient co ntrast perception (Wehner et al. 1969), atypical phototactic resp o nse a n d electroretinogram (Pak et al. 1969; Stark a n d W a s s e r m a n 1972), a s well a s progressive retinal deg ener at i o n (Ambegaokar a n d Jackson 2010). At t h e s a m e time , t h e r e a r e n u m e r o u s whiteeyed lines of Drosophila species t h a t a r e viable a n d u s e d in genetic d a t a , a n d s u b s e q u e n t l y m e r g e d t h e m into a single hybrid assembly. Gene ann o t a t i o n was t h e n carried out on t h e assemb ly, a n d chromosome synteny was m a p p e d , using D. willistoni a s a reference. Base d on t h e genomic information, we utilized CRISPR/ Cas9 to successfully tar get t h e w gene in D. nebulosa. I n d e p e n d e n t white-eyed transgenic flies were t h e n vali dated to as c ertain t h a t w was d i s r u p t e d via nonhomologous end-joining. While responding to phototaxis, we observed t h a t , unlike t h e m a n y other Drosophila stocks with white eyes, w-disrupte d D. nebulosa m a l e s did not a t t e m p t to m a t e with females. The fly species D. nebulosa belongs to t h e willistoni group (Pavan 1946;Schaeffer et al. 2008). This fly h a s b e e n a n attractive s y s t e m to s t u d y t h e evolution of m a t i n g behavior (Spieth 1952;Gleason et al. 2012), cell signaling, gene patterni ng , a n d eggshell morphology (Niepielko et al. 2011(Niepielko et al. , 2014;;Niepielko a n d Yakoby 2014). In D. nebulosa, m a l e fruit flies court by producing a n a n a l droplet a s a nupt i a l gift to t h e female , a n d s u b s e q u e n t l y fannin g it in the ir direction with one wing (Spieth 1952;Steele 1986). Unlike o ther Drosophila species, D. nebulosa requ i r e s vision to locate fem a l es in order to initiate m a t i n g (Spieth 1952;Keesey et al. 2019). In addition, D. nebulosa m a l e s pla ced in c o n s t a n t d a r k n e s s were incapa ble of i n s e m i n a t in g an y f emal e s (Gleason et al. 2012). Since w particip ates in t h e vision process in flies, we hypothesize t h a t de c re a s e d visual acuity, c a u s e d by t h e disruption of w (Xiao et al. 2017) will i m p a i r D. nebulosa ma le s ' ability to recognize potential m a t e s , renderin g t h e m u n a b l e to repr oduce.
As a first s t e p to testing t h e visual r e q u i r e m e n t s underlying m a t i n g success in D. nebulosa on a molecular level, we g e n e r a t e d a high-quality long-read g e n o m e assemb l y using PacBio sequen cing. We produced d e novo preliminar y assemb l i es using 4 differe n t programs, corrected with sh o r t -r e a d Illumina sequencing S u p e r n a t a n t was phenol-chloroform extracted twice, pelleted using isopropanol a n d ethanol, in series, a n d t h e n e l u t e d in nuclease-free water. The gDNA was e v a l uated for quality on a 0.9% agarose gel ( r u n for 45 min a t 100 V) a n d qu ant if ied using a NanoDrop 2000 spec tr oph ot ometer (Thermo Scientific).
Pacific Bioscience single molecule sequ encing (PacBio) was carried out a t t h e W a k s m a n Genomics Core Facility, Rutgers, The S tate University of New Jersey. The DNA was q u a n t i t a t e d usi ng t h e Qubit 2.0 i n s t r u m e n t a n d F r a g m e n t Analyzer with a DNF-467 Genomic DNA 50 kb Analysis Kit according to t h e m a n u f a c t u r e r ' s instructions (Agilent Technologies). S a m p l e s were purified using AMPure XP Clean b e a d s (Agencourt Bioscience Corp., Austi n, TX, USA). Sequencing libraries were constructed following t h e m a n uf a c t u r e r ' s protocol a n d s e q u e n c e d on single-molecule rea l-t ime (SMRT) cells within a PacBio Sequel System, using version 3.0 chemistry a n d 10-h r u n s . Raw r e a d s were g e n e r a t e d by combining o u t p u t s of 4 sequen c ing r u n s , which were carried out using thi s m e t h o d . Reads sh o rter t h a n 3 kb were filtered out using c u t ad a p t v1.8 (Martin 2011).
Genomic DNA was p r e p a r e d for sh o r t -r e a d sequencing, using t h e NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs). The s a m p l e s were s e q u e n c e d a s p a i r e d -e n d 2 100 n t r e a d s on t h e Illumina MiSeq pla tform a t t h e Lewis-Sigler Genomics Core Facility, Princeton University. The FASTQ file was generated , using Illumi na MiSeq Control Software u n d e r def au lt settings. Only pass-filter r e a d s were u s e d for f u r t h e r analysis.
To account for different biases in ge n o m e assemb l ers , preliminary d e novo assemb l i es were constructed using 4 different programs. All assemb ly file n a m e s , with brief descriptions, c a n b e found in S u p p l e m e n t a r y Table 1. First, raw r e a d s were corrected, t r i m m e d , a n d a s s e m b l e d with C a n u v2.1 (Koren et al. 2017(Koren et al. , 2018;;Nurk et al. 2020) def ault p a r a m e t e r s (except -genomeSize ¼ 222m), to g e n e r a t e neb_c. For t h e second preliminary assembly, raw PacBio r e a d s were initially se l f -m a p p e d (setting -x ava-pb), using m i n i m a p 2 to detec t overlaps (Marc¸ais et al. 2018), a n d t h e n co n c at enated into unitigs using m i n i a s m (Li 2016) to g e n e r a t e neb_m1. Raw r e a d s were t h e n m a p p e d back a ga i nst neb_mi using Contiguity a n d quality Assembly statistics (S u p p l e m e n t a r y Table 1) were calculated, using t h e abyss-fac function from ABySS v2.1.5 (Jackman et al. 2017) a n d s t a t s function from BBMap v38.87 (Bushnell 2014). Assembly co mp l eten ess (S u p p l e m e n t a r y Table 2) was e v a l u ate d using BUSCO v5.1.2 (Simao et al. 2015) to co mpare gene content in preliminary a n d composite assemb l ie s to t h e diptera_odb10 lineage d a t a s e t (specifically, -l diptera_odb10 a n d -m genome). The dipter-a_odb10 lineage d a t a s e t s e t contains 3,285 orthologs found to b e p r e s e n t a n d single copy across 56 d i p t e r a n ge n o m e assemb l i e s per fo rmed to date . The presen c e of orthologous g enes in the ir complete form, a n d without duplication, allows u s to a s s e s s how complete our assemb l i es a r e with respec t to gene content. Contiguity a n d co mp leteness of reference Drosophila assemb l i es were a s s e s s e d using t h e s a m e m e t h o d s .
We u s e d t h e Maker v2.31.11 pipeline (Cantarel et al. 2008; m i ni map 2 , generating unpo l ishe d a n d uncorrec ted contig Campbell et al. 2014) to a n n o t a t e t h e polished final composite a ssequen c es, neb_m2. Racon (Vaser et al. 2017) was t h e n u s e d to g e n e r a t e ge n om e co n s e n s u s of t h e uncorrected assemb ly (with i n p u t s <sequences>¼Raw pacbio reads, <overlaps>¼neb_m2, <target sembly (dneb_q3p) (Fig. 4a). For t h e initial r u n , p a r a m e t e r s we edited in t h e maker_opts.ctl d a t a file a s descri bed below, otherwise s e t to 0, or left blank. The polished composite D. nebulosa sequences>¼neb_m1), generating neb_r1. Raw r e a d s were additionassemb ly was u s e d for anno t a t i o n in FASTA f orma t ally m a p p e d a ga inst neb_r1 using m i ni map2 , creating neb_m3.
(<genome>¼neb_q3p, <organism_type>¼eukaryotic). Proteomes Once again, racon was r u n (with i n p u t s <sequences>¼Raw pacbio reads, <overlaps>¼neb_m3, <target sequences>¼neb_r1) to g e n e r a t e t h e final corrected a g e n o me consensus, neb_m. The th i r d assembl y u s e d Flye (Kolmogorov et al. 2019) to a s s e m b l e a n d polish raw r e a d s on def ault settings (except -pacbio-raw), generating neb_f. Finally, raw r e a d s were also a s s e m b l e d using wtdbg2 (Ruan a n d Li 2020) with def aul t settings (except -x sq, -g 222m) to create neb_w1. The final consensus, neb_w, was t h e n g e n e r a t e d using obta i ned from UniProt of species D. melanogaster (GenBank reference: GCA_000001215.4), D. pseudoobscura (GCF_009870125.1), a n d D. willistoni (GCA_000005925.1) were provided a s protein homology evidence in FASTA f ormat (<protein>¼mel_prot, pse_prot, wil_prot). The Repbase r e p e a t library from D. willistoni (Jurka 1998(Jurka , 2000) ) was u s e d a s a m ode l organ is m for soft r e p e a t maski ng (<model_org>¼Drosophila_willistoni, softmask ¼ 1). Ge ne prediction was inferred only using protein homology (<protein2genome ¼ 1). wtpoa-cns (Ruan a n d Li 2020) with def ault settings.
Lastly MAKER behavior settings were s e t (with i n p u t s
Preliminary assemb l i es were t h e n polished usi ng sh o r t -r e a d Illumina sequ enc ing d a t a from D. nebulosa. Short-read d a t a were aligned to e a c h individual preliminary assembl y, using BWA (Li a n d Durbin 2009). The resulting SAM file was converted to t h e BAM format, sorted, a n d indexed using Samtools (Li et al. 2009) with def ault settings. This resulting file, a s well a s its respective preliminary assembly, were t h e n i n p u t into Pilon (Walker et al. 2014) for polishing. P a r a m e t e r s were s e t a s diploid, b u t otherwise kept default. As a note, a "p" was a p p e n d e d to t h e e n d of Pilon-<alt_peptide>¼C, <cpus>¼1, <max_dna_len>¼200,000, <min_contig>¼2,000, <pred_flank>¼200, <AED_threshold>¼1, <split_hit>¼10,000, <tries>¼5).
We r a n MAKER in a Singularity Biocontainer d istri buted by Bioconda (https://bioconda.gi thub.io/). Repeat masking was perfo rmed using RepeatMasker v4.1.1 (Smit et al. 2013(Smit et al. -2015)). Initially, MAKER was u s e d for a b initio gene prediction, a s well a s aligning protein evidence to neb_q3p (with MAKER flags -fix_nucleotides a n d -nodatastore a n d Singularity flags -no-home a n d -cleanenv). MAKER t h e n u s e d 2 gene ann o t a t i o n pr o g r a m s to integ rate evidence a n d produce gene models: SNAP (Korf 2004), a n d corrected preliminary a n d composite D. neb asse m b l i es A u g u s t u s v3.4.0 (Stanke et al. 2008). Both were t h e n t r a i n e d using (e.g. Pilon-corrected neb_w was n a m e d neb_wp) (S u p p l e m e n t a r y Table 1).
Preliminary asse m b li e s were t h e n combined into hybrid assemblies, using quickmerge (Chakraborty et al. 2016) a n d MUMmer (Marc¸ais et al. 2018). This was d o ne by assigning t h e quer y a s s e mbly a s t h e m o s t contiguous, a n d t h e referen ce assemb ly a s t h e t h e r e s u l t a n t predictions, with SNAP specifying a n AED a n d a m i n o acid leng th of 0.25 a n d 50 or greater, respectively (mak-er2zff -x 0.25 -l 50). BUSCO was u s e d to t r a i n A u g u s t u s (with inputs -l diptera_odb10 -m genome -c 30 -augustus -augustus_species flylong -augustus_parameters¼'-progress¼true') on mRNA a n n ot a t e d regions flanked on e ither side by a n additional 1,000 bp.
MAKER was t h e n r e r u n to improve on t h e existing gene m o dels by replacing previous evidence with t h e newly g e n e r a t e d second m o s t contiguous. For example , out of t h e 4 preliminary SNAP a n d A u g u s t u s m od e ls (r e t r a in i n g p a r a m e t e r s ) . assembli es, neb_wp was t h e m o s t contiguous, a n d neb_fp was t h e m o s t complete. As such, the y were selected a s t h e quer y a n d ref-
Additionally, tRNAscan-SE (C han a n d Lowe 2019) was e n a b l e d for t h e detection a n d ann o t a t i o n of tRNAs. The maker_opt s. erence w h e n generating t h e initial composite assemb ly (neb_q1p), ctl file was al t e r e d in t h e following ways: respectively. The specific order of merging is detailed in Fig. 1a.
<protein_gff>¼rnd1.protein2genome.gff, <rm_gff>¼rnd1.repeats. M i n i m u m s e e d contig len gth to b e m e r g e d (length cutoff) was s e t gff, <snaphmm>¼dneb1.l50.aed24.hmm, <est2genome>¼0, to 500. Composite as semb l i es were again polished using sh o r tr e a d Illumina d a t a in Pilon bet w e e n e a c h merge, a s described in t h e previous section. <protein2genome>¼0, < t r n a > ¼ 1 . MAKER was r u n a total of 4 ti mes, e a c h t i m e replacing t h e r e p e a t GFF file a n d SNAP HMM with t h a t of t h e previous r u n . After e a c h iteration, t h e models To account for different biases among genome assemblers, preliminary de novo assemblies were generated using four different programs (rectangles). Preliminary assemblies were polished with Pilon, using short-read Illumina sequencing d a t a from D. nebulosa (circles), a n d th en merged into composite assemblies using quickmerge (diamonds). This was done by assigning the query assembly a s the m ost contiguous, a n d the reference assembly a s the second m o st contiguous. Hollow arrow head s indicate the assembly used a s the query into which the subject assembly was merged. b) Comparison of assembly contiguity. The N50 (top) is the length of the shortest contig a t 50% of the total genome length, while the L50 (bottom) is the numbe r of contigs whose lengths s u m u p to half the genome size. The x-axis shows different versions of the D. nebulosa assembly, n a m e d according to the preliminary or composite assemblies (see Figure 5
A 'p' was appended to the end of Pilon-corrected preliminary a n d composite D. nebulosa assemblies.
were e v al u a t e d for BUSCO comp leteness, n u m b e r of gene models, a n d AED distribution. BUSCO was r u n using t h e trans c ript FASTA a n d A u g u s t u s retraining p a r a m e t e r s of e a c h respective for D. nebulosa orthologs, using protocol descr ibed in section Assigning putative gene function (Campbell et al. 2014). Gene n a m e s were m a p p e d to mode l IDs via Annie (Tate 2014), usi ng t h e afore-MAKER iteration (with inputs -l diptera_odb10 -m transcriptome -c 8 m e n t i o n e d D. melanogaster pro teo m e a n d BLAST results
Dnebulosa -augustus_parameters¼'-progress (S u p p l e m e n t a r y Table 4). Finally, G e n o m e Annotation Generato r ¼true') (S u p p l e m e n t a r y Table 3). Specifically, t h e AED distributi on (S u p p l e m e n t a r y Table 4 a n d Fig. 4e) was calculated using AED_cdf_generator.pl (https: //g ithub . c om/msc ampb e ll / Geno me _ (GAG) (Geib et al. 2018) was u s e d to r e n a m e gene models, a s well a s to pull ann o t a t i o n statistics. Drosophila nebulosa gene mode ls were protein-aligned , via blastp, a ga inst t h e D. willistoni pr oteo me annotation/blob /master/AED_cdf_generator.pl), by using t h e a s a n a d d it ion al m e a s u r e of or t h olog h om ology. m a s t e r GFF file a s t h e inpu t , a n d specifying t h e bin size (-b 0.025).
Gene model IDs were r e n a m e d a n d m a p p e d us ing MAKER's maker_map_ids, map_gff_ids, a n d map_fasta_ids functions, following a protocol described in t h e section Renaming Genes for GenBank Submission in Campbell et al. (2014). The pr o teo me of D. melanogaster was u s e d a s a BLAST reference to obtain n a m e s
We u s e d t h e BUSCO_phylogenomics pipeline (McGowan et al. 2020; McGowan a n d Fitzpatrick 2020) to a s s e s s t h e phylogenomic position of our D. nebulosa assemb ly with respec t to re l a t e d species with ge n o m e assemb l i es available. This pipeline involves first r u n n i n g BUSCO on an y g e n o m e s to b e included in t h e phylogeny to find orthologous genes for phylogenomic t r e e building. Using t h e s a m e BUSCO settings described above, we analyzed 26 ge n om e assemb l i es from 23 species (S u p p l e m e n t a r y Table 8) (Clark et al. 2007;Zimin et al. 2008;Hoskins et al. 2015;Chakraborty et al. 2017;Zhang, Yu, et al. 2018;Liao et al. 2019;Paris et al. 2020; P i n h a r a n d a et al. 2020; Reilly et al. 2020). Assembli es from t h e D. willistoni a n d D. saltans subg ro ups were also included to e n s u r e a d e q u a t e phylogenomic comp arison within a n d to ad j a c e n t monophyletic b r a n c h e s (Kim et al. 2020).
functio n, to calculate t h e n u m b e r of genes within e ac h 10 kb window . Statistics were visualized for t h e largest 11 scaffolds using t h e circlize package (Gu et al. 2014) in RStudio.
To develop a white-eyed Cas9-expressing transgenic D. nebulosa fly, we u s e d homology-directed repai r to d i s r u p t w, while simultaneously inserting Cas9 u n d e r a nanos prom oter. The CRISPR/ Cas9 s y s t e m works by creating a d o u b le -s t r and break proximal to a specified 20 bp tar g et site ad j ac e n t to a protospacer ad j a c e n t Results for e a c h BUSCO r u n were t h e n u s e d by t h e motif (PAM) sequ en ce , facilitated by a guide p l a s m i d (Gratz et al. BUSCO_phylogenomics pipeline to create t r i m m e d a l i g n m e n t s for e a c h gene t h a t was p r e s e n t a n d single copy in every quer i e d genome. Alignments were per f ormed using MUSCLE v 3.8.31 (Edgar 2004a, 2004b) a n d t r i m m e d usi ng TrimAl v 1.4 (Capella-Gutierrez et al. 2009). These a l i g n m e n t s were t h e n u s e d to infer phylogenies by e i ther concatenation a n d phylogenetic analysis in IQ-Tree v1.2.12 (Nguyen et al. 2015) ( supermat r i x m e t h o d ) or by inferring individual gene t r e e s with IQ-TREE a n d performing species t r e e inference usi ng ASTRAL v. 5.7.7 (Zhang, Rabiee, et al. 2018) (s u p e r t r e e meth od) . We r a n BUSCO_phylogenomics with def aul t settings, except t h a t IQ-TREE (Nguyen et al. 2015) was r u n using t h e -safe flag. All t r e e s were rooted, using Scaptodrosophila lebanonensis a n d visualized using iTOL (Letunic a n d Bork 2021). Chromosome synteny We ne x t placed D. nebulosa scaffolds to predicted chr omo so mes by comparing conserved loci to a reference genome. D. willistoni was chosen a s a refer ence d u e to t h e genome's similar chromosomal a r r a n g e m e n t to D. nebulosa. As a note, t h e D. willistoni caf1 (GCA_000005925.1) a n d D. willistoni 17 (GCA_018903445.1) assembli es will hereby b e referen ced a s wil_caf1, a n d wil_17, respectively. First, we identified chromosomal locations of wil_caf1 2013). Point m u t a t i o n w alleles in D. melanogaster from Mackenzie et al. (1999) were m a p p e d to exons 3-6 in D. nebulosa. The locus was tar g e t e d (Fig. 6a) by aligning bo th s e q u e n c e s in MEGA ( K u m a r et al. 2016) a n d choosing PAM sites which flank t h e predicted region. Target cut sites were d e t e r m i n e d usi ng CRISPR Optimal Target Finder (Gratz et al. 2013), a n d cleavage efficiency was predicted usi ng CRISPR Efficiency Predictor (Housden et al. 2015). The formerly m e n t i o n e d pr o gr a m was u s e d to find tar g et s e q u e n c e s ad j a c e n t to PAM sites, a n d to c o mp are t h e m to a reference ge n o m e to look for similar off-target cut sites. Since t h e D. nebulosa g e n o m e was not listed on t h e site, we a t t e m p t e d to account for off-target cut sites using t h e D. willistoni geno me. The selected tar g et s e q u e n c e s were t h e n aligned via BLASTn to our D. nebulosa g e n o m e to look for m a t c h e s .
To repai r t h e d o u b l e-strand break(s) by t h e guide(s), a donor vector was designed featuring t h e Cas9 gene u n d e r t h e nanos promoter (see CRISPR constructs section in Materials and methods). The vector insert was flanked by two 1,000 bp, a r m s which a r e homologous to t h e D. nebulosa w loci surr oun di ng t h e tar get region, a s described in Gratz et al. (2013). Three s e p a r a t e guide injections (Rainbow Transgenics, CA) (Fig. 6c) were u s e d increase t h e likelihood of a u n i q u e target, a s well a s to t h e t e s t t h e efficiency of 1 assemb l y scaffolds, using d a t a from previous stud ie s vs 2 guides. All injections included t h e Cas9-containing donor (S u p p l e m e n t a r y Table 9) (Schaeffer et al. 2008;Garcia et al. 2015). In a n a t t e m p t to u s e a m o r e contiguous reference, we u s e d S a t s u m a 2 (https://github.co m/bioinfologics/satsuma2) (Grabherr p l a s m i d (1.12 lg/ll) a n d Cas9 protein (5 lg/ll) (ThermoFisher #A36498). Injections differed in t h e combination of guide plasmids, where injection #1 contained t h e neb_w_guide1 p l a s m i d et al. 2010) to find syntenic scaffolds bet w e e n t h e wil_caf1 a n d (2.58 lg/ll), injection #2 contai ned neb_w_guide2 p l a s m i d wil_17 assemb l i es (S u p p l e m e n t a r y Table 10). This was d one spe-(1.33 lg/ll), a n d injection #3 contai ned neb_w_guide1 a n d cifically by comparing only c h r o m o s o m e -a n n o t a t e d wil_caf1 scaffolds to t h e 20 largest wil_17 scaffolds. From th is output, wil_17 scaffolds syntenic to th o s e of wil_caf1 were selected, r e n a m e d according to chr om oso me (S u p p l e m e n t a r y Table 11), a n d t h e n co m p a r e d with 11 largest D. nebulosa scaffolds using S a t s u m a 2 (S u p p l e m e n t a r y Table 12). As a n additiona l evaluation, a n n ot a t e d wil_caf1 scaffolds were also c o m p a r e d with t h e 11 largest D. nebulosa scaffolds using S a t s u m a 2 (S u p p l e m e n t a r y Table 13). All S a t s u m a 2 compariso ns were r u n with defau lt settings. The R circlize package was u s e d to visualize chromoso me synten y betw een D. nebulosa a n d D. willistoni, usi ng a repre s e n t a t iv e s u b s e t of a l i g n m e n t s with identities greater t h a n 0.75. Only D. willistoni scaffolds with over 2,000 aligned regions with D. nebulosa were visualized (Fig. 5 a n d S u p p l e m e n t a r y Fig. 1).
Transposable e l e m e n t density was m a p p e d to t h e 11 largest scaffolds to predict centromeric locatio ns. To m a p transp o sab le el em en ts in t h e D. nebulosa geno me, we u s e d t h e Bedto ols v2.50.0 (Quinlan a n d Hall 2010) makew indow s function to div ide t h e largest 11 scaffold len gths in t h e assem bly into 10 kb w indow s (setting -w 10000 -s 10000), generating neb_win.10k. We t h e n u s e d LINE, LTR, DNA transposa bl e el em en ts , a n d helitron MAKER a n n o t a te d repeats , a s well a s neb_win.10k, a s i npu ts for t h e Bedtoo ls coverage neb_w_guide2 (1.76 lg/ll) (Fig. 6c). All oligonucleotides a r e shown in S u p p l e m e n t a r y Table 14.
Two different guide oligonucleotides were ligated into individual pU6-BbsI-chiRNA p l a s m i d s (Melissa Harrison, Kate O'Connor-Giles, a n d Jill Wildonger; Addgene p l a s m i d # 45946), a s described in Gratz et al. (2013). The donor vector (Fig. 6a) was designed using a modified Gratz et al. (2013) procedure. Left a n d right homology a r m s were amplified from genomic DNA of whole D. nebulosa flies. The donor vector backbone a n d nos-Cas9 locus were amplified from a pnos-Cas9-nos p l a s m i d (Addgene p l a s m i d # 62208) (Port et al. 2014). A comple te list of p r i m e r s u s e d is listed in S u p p l e m e n t a r y Table 14 u n d e r Primers used for CRISPR.
All donor vector f r a g m e n t s were ligated into a circular p l a s m i d via New England Biolabs HiFi Assembly Master Mix Gibson Assembly (E2621). The 4-fragment Gibson assemb ly u s e d a 1:1 vector: insert ratio, containing 148.85 ng pnos-Cas9-nos backbone (28. g m e n t 3 for 15 min. To limit t h e bri g htness ev en further, t h e light was covered by one layer of a p a p e r towel. The q u a n t i t i e s of flies in e a c h s e g m e n t were again recorded. Four trials were conducted for b o th wildtype, a s well a s white-eyed m a l e D. nebulosa. The e x p e r i m e n t was carried out a t roo m t e m p e r a t u r e (23C). The e x per i menta l s e t u p is visualized in Fig. 7a. Results were analyz ed for significance in t h e d a t a s e t , using 1-way ANOVA, a n d s u b s e q u e n t Tukey HSD t e s t s for all-verses-all comparison of t r e a t m e n t m e a n s were performed.
Sequencing a n d d e novo g eno m e assemb ly
Long-read sequencing g en era ted 13 Gb of s eq uen ce fro m 1,717,740 s ub re a d s above 3 kb with a r e a d N5 0 of 8.4 kb. Short-read Illumina sequencing g en er ate d 29,211,787 forward reads, 10 0 bp in length.
To account for different biases in gen om e a ss em bl ers we u se d 4 s ep a r at e pr o grams to gen er ate preliminary assemblies, a n d sub sequ en tl y m er g ed t h e m in a step-wise fashion (Fig. 1a). Co mposite assembly neb_q1p (merge of neb_wp a n d neb_fp) show ed considerable im pr o vem en ts in contiguity, a s w ell a s completeness similar to neb_fp. S u b s eq u e n t m er g es w ith neb_cp, a n d t h e n neb_mp, impro ved t h e r es u l ta nt composite ass embl ies (neb_q2p a n d neb_q3p, respectively), th o ugh only marginally (Fig. 1b). Contiguity of neb_q3p also compar es favorably with other available Drosophila assemblies in t h e willistoni gro up (Fig. 2). The final assem bly (neb_q3p) h a s a total of 1,600 scaffolds, w ith a n N5 0 of 20 .9 Mb, a n d a total
D. simulans D. sechellia D. melanogaster D. erecta D. yakuba D. suzukii D. ananassae D. pseudoobscura D. persimilis D. willistoni caf1 D. willistoni.17 D. willistoni.00 D. paulistorum.L06 D. paulistorum.L12 D. tropicalis D. insularis D. nebulosa D. sucinea D. saltans D. prosaltans D. neocordata D. sturtevanti D. virilis D. mojavanesis D. grimshawi S. lebanonensis 0 10 20 30 N50 (Mb) Fig. 2. The D. nebulosa assembly is highly contiguous compared with other available Drosophila assemblies. Shown is a phylogenomic tree of D. nebulosa a n d Drosophila assemblies referenced in this work, with associated N50 values. The phylogenomic tree of the Drosophila genus shown is based on supermatrix methods. The topology was inferred via concatenation of 3285 Universal Single Copy Orthologs present in all lineages a n d rooted with Scaptodrosophila lebanonensis. Bootstrap values were 100% for all branches. Light a n d dark shaded species are members of the willistoni group a n d bocainensis subgroup, respectively. (a) Protein evidence D. melanogaster Genome assembly neb_q3p (b) (c) 12 3 10 2.5 D. pseudoobscura 8 D. willistoni 6 M M M M 4 2 2 1.5 1 0.5 Gene models 1 (d) Gene models 2 Gene models 3 Gene models 4 0 1 2 3 4 Annotaon iteraon (e) 0 1 2 3 4 Annotaon iteraon 1 4 C: 3006 [S: 2965, D: 41], F: 67, M: 212, n: 3285 0.9 0.8 3 C: 3031 [S: 3014, D: 17], F: 60, M: 194, n: 3285 0.7 0.6 2 C: 3040 [S: 3023, D: 17], F: 57, M: 188, n: 3285 0.5 0.4 1 C: 3036 [S: 3019, D: 17], F: 61, M: 188, n: 3285 0.3 0.2 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0.1 BUSCOs 0 Iteraon 1 Iteraon 2 Iteraon 3 Iteraon 4 Complete and single-copy Complete and duplicated Fragmented Missing Annotaon edit distance size of 177 Mb. Statistics fo r ea ch preliminary a n d composite assembly a r e listed in Supp lem en ta ry
Table 1. Analysis of 3,285 universal single copy Dipteran orthologs (BUSCO diptera_odb10 da ta se t) in neb_q3p revealed 98 .2% (3,229) to be p r e s en t a n d full length (97.7%, 3,211 single-copy; 0.5%, 18 duplicated), 0.9% (2 8) were p re s e nt b u t fragmented, a n d 0.9% (28) of t h es e genes were
missing fro m our assembly (Fig. 3). Co mparative BUSCO scores for our preliminary a n d final co mposite ass embl ies a r e listed in Sup pl em en ta ry Table 2.
Gene mode l s were r e t r a i n e d via t h e MAKER pipeline a total of 4 t i m e s (Fig. 4a). Since t h e fourth r u n of t h e pipeline produ ced little models, BUSCO scores, a n d a n n o t a t i o n edit di stanc e (AED) (Fig. 4, b a n d e; S u p p l e m e n t a r y
Table 3), t h e th i r d iteration of gene predictions was chosen a s t h e final a n n o t a t i o n a n d will t h u s b e repo rted on in th i s section. Ge n o m e ann o t a t i o n thr o u g h d e novo prediction a n d homology with D. melanogaster produc ed 13,067 gene mo de ls (Table 1). Protein BLAST a l i g n m e n t s of D. nebulosa model s with D. melanogaster a n d D. willistoni g e n e r a t e d 12,548 a n d 12,578 al i gnments, respectively, indicating orthology with b oth species (S u p p l e m e n t a r y Table 4). BUSCO analysis of t h e transc r ip t o me revealed 92.5% (3,040) completed (92.0%, 3,023 singlecopy; 0.5%, 17 duplicated), 1.7% (57) fr a g m e n t e d , a n d 5.8% (188) missing orthologs (Fig. 4d) suggesting t h a t our ann o t a t i o n includes t h e vast majority of g enes p r e s e n t in our assembly. AED, a m e a s u r e m e n t of how well a n a n n o t a t i o n ag rees with overlapping protein homology evidence (scores 0 a n d 1, deno ting perfect a n d no a g r e e m e n t to aligned evidence, respectively) (Holt a n d Yandell 2011), shows 97% of t h e ann o t a t i o n with a score of 0.5 a n d u n d e r (Fig. 4e; S u p p l e m e n t a r y Table 5). D. nebulosa (Pita et al. 2014), c a n b e found on scaffold dneb_sca_3 in our assembly. The X chro m o so me a p p e a r s to b e less contiguous with dneb_sca_2, 5, a n d 9 syntenic to t h e D. willistoni left a r m (ChrXL), a n d dneb_sca_2, 4, 7, a n d 8 syntenic to t h e D. willistoni right a r m (ChrXR1-4). Of note, dneb_sca_2 a p p e a r s to s p a n across b oth a r m s of D. willistoni Chr omoso me X, with t h e right a r m syntenic to position 22 ,716-8,714,232, a n d left to 8,775,313-21,991,806. Syntenic a l i g n m e n t of D. nebulosa a n d D. willistoni (wil_caf1) assemb l ie s (S u p p l e m e n t a r y Fig. 1) were largely consistent with t h e a fo rementi o ned d a t a , with t h e exception of a r e a rr a n g e m e n t bet w e en t h e right a r m of chr omoso mes X a n d 2, which differ slightly in position a n d size.
As a m e t h o d of deter m i n i n g potential ce n t r o m e r e location within t h e assemb ly, we m a p p e d all a n n o t a t e d class I a n d II transp o sabl e e l e m e n t s to t h e largest 11 scaffolds. We s e e considerable e n r i c h m e n t of transp o s a b le e l e m e n t s a t t h e s t a r t of dneb_sca_3 a n d 9, a n d a t t h e e n d of dneb_sca_0, 1, a n d 4 (Fig. 5). A few s ma ll er spikes of trans p o s a b le e l e m e n t s a r e i n t e r s p e r s e d thro u g h out t h e scaffolds, however, t h e higher density regions a t t h e scaffold e n d s indicate t h e s e locations a s likely centromeres . white w a s successfully di s r u p t e d in D. nebulosa, b u t not repaired via homology-directed repair Following g e n o m e assemb l y a n d gene annotat io n, we a i m e d to t e s t t h e r e q u i r e m e n t of vision in D. nebulosa m a t i n g by generating a white-eyed D. nebulosa, a s a proof of concept. If m u t a t i o n in w c a n b e tolerated, a s in othe r species, we i n t e n d e d to insert t h e Cas9 gene into t h e w gene to obtain a stock t h a t c a n potentially b e u s e d for f u t u r e CRISPR/Cas9 ge n o m e engineering. Briefly, t h e D. nebulosa w locus was tar g e t e d by a combination of 2 guides, Cas9 protein, a n d a homology directed repai r vector with t h e Cas9 gene (more details c a n b e found in t h e Materials and methods)
Number of genes 13,067 Number of exons 52,709 Number of introns 39,642 Number of CDS 12,698 Overlapping genes 94 Contained genes 43 Total gene length 39,234,692 bp Total exon length 21,059,387 bp Total intron length 18,254,589 bp Total CDS length 21,016,413 bp Shortest gene 68 bp Shortest exon 3 bp Shortest intron 8 bp Shortest CDS 84 bp Longest gene 88,781 bp Longest exon 13,185 bp Longest intron 21,319 bp Longest CDS 68,394 bp Mean gene length 3,003 bp Mean exon length 400 bp Mean intron length 460 bp Mean CDS length 1,655 bp % of genome covered by genes 22.2 % of genome covered by CDS 11.9 Mean mRNAs per gene 1 Mean exons per mRNA 4 Mean introns per mRNA 3
(Fig. 6a). Since t h e w gene is on t h e X chromosome, t h e expectation was to obta in white-eyed m a l e flies hemizygous for d i sr u p t e d gene. In total, 13 F1 lines of white-eyed D. nebulosa m a l e s were selected positive for t h e CRISPR disruption (Fig. 6, b a n d c).
Despite r e p e a t e d a t t e m p t s , fem a l es r e m a i n e d heterozygous for t h e w null allele, t h u s t h e white-eye phen o t yp e was only found in ma les .
Although t h e initial i n t e n t was to in s e r t t h e Cas9 gene into t h e w gene, PCR validation of white-eyed D. nebulosa CRISPR tar get loci revealed t h a t n e i t h e r insert integration, nor complete deletion occurred in a n y of t h e lines (Fig. 6, c-h). Interestingly, lines successful for disruption of w were all from embryos injected with b o th guide p l a s m i d s (Fig. 6c). While t h e 2 guides pr o mo te d gene disruption, deletions were only found to b e p r e s e n t a r o u n d one PAM site p e r line (7 out of t h e total of 10 achieved from guide 1, Fig. 6g), b u t never a t both. Instead , b oth guide p l a s m i d s c reated asynchronous 1-14 bp deletions on or u p s t r e a m of t h e PAM sites (Fig. 6, d-h). In one case, a 565-bp deletion was characterized adj a c ent to guide 1 (Fig. 6f).
white-disrupted D. nebulosa m a l e s resp o n d to light b u t w e r e not observed courting femal e s A cross bet w e e n wild-type f ema l es a n d white-eyed m a l e s failed to produce developing embryos. As a result, white-eyed f em a l es were nev er observed in a n y of t h e lines. This is s u p p o r t e d by t h e fact t h a t a white-eyed m a l e in th is cross was no t observed courting t h e wild-type f emal e even once over a period of 4 h. At t h e s a m e time, a control cross of a wild-type m a l e a n d f emal e displayed 13 in s t a n c e s of distinctive courtship. The courtship was observed in varying intervals (42.0 6 30.6 s) for 9.1 min, cumulatively. To a s s e s s phototaxis in wild-type a n d white-eyed D. nebulosa, we placed flies in a plastic t u b e s e g m e n t e d into 3, a n d quanti fied t h e average n u m b e r of flies in e a c h s e g m e n t of t h e t u b e after dark a n d light conditions. All flies were initially placed in t h e proximal e n d of s e g m e n t 1, a n d a light source was placed facing t h e distal e n d of s e g m e n t 3 (Fig. 7a). We expected t h a t flies kept in d a r k n e s s would not show a ten den cy to travel to an y specific p a r t of t h e tu be . Consequently, t h e distribution of t h e fly popula- ). Hence, D. nebulosa h a s 3 chromosomes: X, 2, a n d 3; with t h e X a n d 2 consisting of a left a n d right a r m s (Pavan 1946;Valente et al. 1996;Schaeffer et al. 2008). As a n a t t e m p t to correlate s o m e of t h e larger scaffolds to the i r potential chromosome, we s e a r c h e d for syntenic regions bet w e e n D. nebulosa a n d D. willistoni assemb l i e s (Fig. 5). Altogether, th i s evidence suggests t h a t t h e D. nebulosa assemb ly succeeded in generating scaffolds t h a t a r e con gruent with known D. willistoni chromosomal a r m s 2L (dneb_sca_0), 2R (dneb_sca_1), 3 (dneb_sca_3, 6, a n d 10), XL (dneb_sca_2, 5, a n d 9), a n d XR (dneb_sca_2, 4, 7, a n d 8). These d a t a show t h a t t h e largest 11 scaffolds from t h e assemb l y account for all 3 D. nebulosa chrom osomes (5 chro m o so me arms ). Additionally, t h e ey, ci, a n d Ank genes a r e all on sca_3 in t h e D. nebulosa assembly. This reflects t h e fusion of t h e dot chro mosome a n d chromosome 3 a n d is in s e g m e n t af ter 15 min of dark conditions (Fig. 7b; S u p p l e m e n t a r y a g r e e m e n t with e xp e r im e n t a l result s of previous stud i e s Table 6). This indicated t h a t b oth wild-type a n d white-eyed D. nebulosa m a l e s resp o n d to light.
In order to g e n e r a t e tools for genetic a n d genomic ana l y ses in D. nebulosa, we a s s e m b l e d a long-read a n n o t a t e d D. nebulosa genome. This assemb l y is highly complete a n d contiguous a n d co m pares favorably to o ther ge n o me assemb l i e s in t h e willistoni clade. Using t h e ge n o m e of D. willistoni a s a reference, we predicted t h a t n e a r -e n t i r e chromosomal a r m s c a n b e reconstruct ed with 1-4 scaffolds from t h e D. nebulosa assembly. Scaffolds also s p a n intergenic regions, facilitating t h e design of molecular e x p e r i m e n t s within t h e species. In particular, t h e asse m b l y contiguity was ou t s t a n d i n g relative to m a n y available Drosophila gen ome assemb li es. Comparison of N50 values across 26 Drosophila assemb l i es shows our assemb l y a s t h e highest of t h e species s a m p l e d within its clade, with a contig size compara b le to t h e c u r r e n t D. melanogaster assemb l y (Fig. 2). Assembly co mpl e teness was e v a l uate d via BUSCO, with D. nebulosa showing scores in line with t h e o ther assemb l y references (Fig. 3; S u p p l e m e n t a r y Fig. 8).
Annotation BUSCO scores a r e co mparab le to t h a t of t h e genome, indicating t h a t our ann o t a t i o n likely c a p t u r e s t h e majority of protein coding genes in th i s species. The n u m b e r of D. nebulosa genes (Table 1) a n n o t a t e d in t h e final version (iteration 3) is comparable to t h e n u m b e r of D. willistoni protein coding g enes in t h e c u r r e n t assemb ly ann o t a t i o n (GCF_000005925.1) (Clark et al. 2007;Zimin et al. 2008), which is expected for 2 species of t h e s a m e subgroup. This compariso n provides f u r t h e r confidence in our d e novo assemb ly.
Phylogenomic t r e e s inferred using s u p e r m a t r i x (Fig. 2) a n d s u p e r t r e e appr o a ch e s (S u p p l e m e n t a r y Fig. 2) recovered identical t r e e topologies a n d placed D. nebulosa a s sister to a clade containing D. willistoni, D. paulistorum, D. tropicalis a n d D. insularis, a n d within t h e monophyletic willistoni group (van d e r Linde a n d Houle 2008). We recover a no ns ister relationshi p bet w e e n D. nebulosa a n d D. sucinea. This finding s u p p o r t s previous work suggesting paraphyly of t h e bocainensis subg ro up (Gleason et al. 1998;Tarrio et al. 2000;Zanini et al. 2018).
In t h e several species of t h e willistoni group, t h e dot chrom os o m e does not exist alone a n d is i n s t e a d fus ed to chro m oso me 3 (Papaceit a n d J u a n 1998; Pita et al. 2014). Scaffolds constituting t h e X chr om oso me a p p e a r to b e less contiguous, m o s t likely d u e to homologous, b u t divergent X a n d Y gametologs from t h e mix of m a l e a n d fem a le D. nebulosa u s e d for sequencing. As of note, t h e a s s i g n m e n t of D. willistoni chr om oso me 2 a r m s h a v e b e e n d eb a t e d (Rohde et al. 1995;Schaeffer et al. 2008;Garcia et al. 2015), b u t for t h e p u r p o s e s of th is discussion, t h e stud y by Garcia et al. (2015) was mainly referenced. Accordingly, it sho uld b e n o t e d t h a t assigning scaffolds to chr om osomes is b a s e d on sy nteny with D. willistoni.
The ge n o m e of D. nebulosa h a s long b e e n esta b l i s h e d to contain m a n y i n s t a n c e s of chrom osomal r e a r r a n g e m e n t s (Pavan 1946;Vale nte et al. 1996; Papaceit a n d J u a n 1998; Pita et al. 2014). In addition, genetic recombination in t h e X chrom oso me is m o r e f r e q u e n t t h a n in a u t o s o m e s (Rius et al. 2016). This m a y account for t h e syntenic variation we s e e in reference to t h e D. willistoni assemblies , s u c h a s t h e r e a r r a n g e m e n t of specific regions betw een chr om oso me a r m s XL a n d XR, or XR a n d 2R (Fig. 5). Although th is variation could b e d u e to contig mis-joining in t h e reference assemb li es, interspeci es chromosomal variation h a s b e e n previously character ized in D. willistoni (Rohde a n d Vale nte 2012). Overall, t h e l atte r possibility is favored since b oth reference D. willistoni assemb li e s (wil_caf1 a n d wil_17) a r e from different isolates (S u p p l e m e n t a r y Table 8).
As a final metric of assemb l y contiguity a n d completen ess, we s e t to a s s e s s how well scaffolds c a n rec apitul ate D. nebulosa chromosomal a r m s . One m e a s u r e of th i s is w h e t h e r t h e assemb l y scaffolds include centromeric regions a t t h e e n d of t h e chromosome. In drosophilids, transp o s a b l e e l e m e n t s h a v e b e e n shown to b e d i stribute d m o r e densely in centro-a n d telomeric regions, a s well a s other regions of low recombination r a t e (Thomas et al. 2015;Rius et al. 2016). Indeed, we s e e high density regions of transp o s a b l e e l e m e n t s a t t h e e n d s of 5 D. nebulosa scaffolds t h a t a r e predicted to e a c h comprise different chro mo so me a r m s (Fig. 5). F urtherm o r e , MAKER r e p e a t a n n o t a t i o n did no t show a high density of HeT-A, TART, or TAHRE retrotransp o sab l e e l ements , which a r e known to constitute Drosophilid telomeres. Altogether, it provides evidence t h a t scaffolds include s e q u e n c e u p to centromeric regions.
Using CRISPR/Cas9 g e n o m e editing h a s a great potential to develop ne w a n d powerful mode l or gan isms to a d d r e s s evolution-(fusion of Muller e l e m e n t s E þ F). Previous stud i e s h a v e u s e d fluo-ary processes re l a t e d to cell signaling, t is s u e pa ttern in g, rescence in s itu hybridization to d e m o n s t r a t e t h a t t h e ey, ci, a n d Ankyrin (Ank) genes, which a r e p r e s e n t on chro mo some 4 in D.
morphogenesis, a n d behavior. In addition, it would b ypass years of m u t a t i o n -i n d u c e d screens, a s was d o ne for m a n y alleles found in D. melanogaster. In D. melanogaster, white-eyed flies a r e commonly u s e d for transgenic e xper i ments. To our knowledge, th i s is t h e first t i m e CRISPR/Cas9 was successfully utilized in D. nebulosa by targeting w in t h e ge n o m e (Fig. 6b). At t h e s a m e time, characterization of t h e d i s r u p t e d locus revealed t h a t al tho u gh flies co m p a r e d single crosses of a white-eyed a n d wild-type D. nebulosa m a l e pa i r e d with a virgin female . A clear difference in courtship display was preval ent bet w e e n t h e crosses of wild-type D. nebulosa, where m a l e s frequentl y a t t e m p t e d courting f ema l es a n d exploring t h e vial. This observation is in co ntrast to t h e white-ey ed injected with b oth guides were positive for d i s r u p t e d w, only one ma les , which did no t a t t e m p t a n y courting, even w h e n of t h e 2 PAM tar g e t s was cut in every case (Fig. 6c). Interestingly, using b o th guide RNAs g e n e r a t e d 9 different deletions in t h e gene (Fig. 6, g a n d h). This strategy c a n potentially serve a s a tool to g e n e r a t e different alleles a n d let selection act on a viability scale. While t h e s e were different types of deletions, n o n e could produce a viable white-eyed f em a l e fly.
Short indels proximal to t h e tar g e t e d PAM sites a r e indicative of nonhomologous e n d joining, a s oppo sed to homology-directed repai r (Gratz et al. 2013). This is f u r t h e r s u p p o r t e d by t h e fact t h a t t h e nos-Cas9 cassette, designed to inte grate within t h e w gene, was not p r e s e n t in a n y of t h e t e s t e d lines (Fig. 6, d-f). The role of t h e donor vector is to repai r double s t r a n d brea ks c reated by Cas9. One possible r e a s o n for th i s is t h a t t h e homology-directed repai r pa th w ay is known to b e less efficient t h a n t h e nonhomologous e n d joining (Roy et al. 2018). Therefore, breaks in t h e ge n o m e m a y h a v e b e e n ligated together before t h e donor vector was able to repai r t h e m . Previous s t u d i e s h a v e u s e d various m e t h o d s to increase CRISPR efficiency, s u c h a s piggyBac-mediated integration of t h e nos-Cas9 locus (Gratz et al. 2014; Nishizawa-Yokoi a n d Toki a p p r o a c h e d by females. In fact, t h e s e m a l e s rarely move a t all in t h e vials. In light of t h e s e observations, a n d t h e estab li shed m a ti n g behavior of D. nebulosa, it is possible t h a t white-eyed m a l e s a r e u n a b l e to visually locate t h e female.
One way to a s s e s s t h e e x t e n t of lowered visual acuity is to e x a m i n e t h e effects of t h e d i s r u p t e d w gene on phototactic respo nse in D. nebulosa. The expectation would b e t h a t wild-type D. nebulosa capable of perceiving light would b e a t t r a c t e d to it a n d cluster n e a r t h e source. Conversely, D. nebulosa t h a t a r e completely blind would b e expected to b e ignorant to t h e light source, a n d t h u s d i sperse rando mly. F urtherm or e , phototactic success in white-eyed flies d u e to perceptio n via ocelli is also unlikely, since w is requ i r e d for p ig mentat io n in t h e eyes, a s well a s t h e ocelli (Levis et al. 1985;Caldwell et al. 2007). Our findings showed t h a t similarly to wild-type D. nebulosa, white-eyed flies were a t t r a c t e d to light a n d traveled toward t h e source (Fig. 7b). These Several Drosophila species a r e available a s viable white-eyed stocks a n d u s e d in transgenic e x p e r i m e n t s (Holtzman et al. 2010;S tern et al. 2017). However, m o s t of t h e s e species court via acoustic, chemical, a n d tactical modalities a n d a r e not solely d e p e nd e n t on vision. In contrast, m a t i n g in several species h a s b e e n found to requ ir e vision (Jezovit et al. 2017;Keesey et al. 2019). For example, D. nebulosa m a l e s initiate courtship by upper cu t t i ng t h e f ema le with th e ir legs, st a n d i n g perpend icularly a n d angling the i r posterior toward her, a n d silently fanni n g a n e x t r u d e d a n a l droplet in h e r direction via flicking motions with one wing. In t h e absenc e of light, D. nebulosa m a l e s were u n a b l e to orient t h e m s e l v e s toward t h e female , resulting copulation failure (Gleason et al. 2012). In species s u c h a s D. nebulosa a n d o thers like it, we expected t h a t impairing visual acuity a n d optical insulation thro u g h disruption of w (Ferreiro et al. 2017) would jeopardize courtship rituals, a n d t h u s copulation. While commonly use d , white-eyed D. melanogaster d e m o n s t r a t e d redu c e d courtship. The phen o ty p e was shown to b e alleviated in t h e s e flies with t h e introduction of t h e mini-w gene (Xiao et al. 2017). It is possible t h a t copulation was still successful in white-eyed D. melanogaster, since m a l e s court using wing-vibrations to produce a speciesspecific "song" (Spieth 1952). Conversely, disruption of w in D. suzukii, whose courtship rituals a r e m o r e similar to D. nebulosa, resu l t e d in no a t t e m p t s a t courtship or copulation (Yan et al. 2020).
In our study, pairing white-eyed m a l e s with virgin wild-type D. The D. nebulosa species provides a compelling model s y s t e m to investigate a variety of biological p h e n o m e n a , s u c h a s evolution of cell signaling, patterni ng, morphology (Niepielko et al. 2012), chromosomal a r r a n g e m e n t s (Valente et al. 1996), m a t i n g behavior (Gleason et al. 2012), a n d ev en radiation resistan ce (Kratz 1975). Here we suggest t h a t , given t h e f u n d a m e n t a l evolutionary differences in D. nebulosa's courtship, th is species is a n attractive organism to develop genetic tools to stud y t h e visual r e q u i r em e n t s underlying m a t i n g success. Unlike t h e challenges to r e a r D. willistoni (Holtzman et al. 2010), D. nebulosa is simp le to r e a r in t h e s a m e conditions a s D. melanogaster. However, t h e disruption of t h e visual syste m , which is requ i r e d for mati ng, should b e avoided, a n d othe r phenotypic m a r k e r s shou ld b e considered t h a t a r e not involved with vision. A potential solution is to choose a selectable m a r k e r which is not involved in vision a n d behavior. One s u c h possibility is t h e wing mar k er, crossveinless (cv) fCG12410, FBgn0000394g. This gene is known to b e on t h e X chromosome in D. melanogaster. In our D. nebulosa assembly, cv h a s CDS length of 633 bp, a n d is found on dneb_sca_5 (predicted to belong to t h e chromosomal a r m XL). The gene's sma ll size would m a k e it easi er to clone into vectors for phenotypic res cue (Shimmi et al. 2005), a n d like white, sex-linkage would allow u s to sc reen for m a l e s of t h e F1 generation.
Data availability nebulosa fema le s produce d eggs, b u t neve r a n y larva. This Whole G e no m e Shotgun project h a s b e e n dep osited a t DDBJ/ Consequently, white-eyed femal es were never observed in a n y of t h e lines. We predicted t h a t m a l e a n d fem a le crosses failed to copulate, t h u s leading to unfertilized eggs. To s u p p o r t this, we ENA/GenBank u n d e r t h e accession JANFPS000000000. The version described in th is p a p e r is version JANFPS010000000 S u p p l e m e n t a l m a t e r i a l is available a t G3 online.
A c k n o w l e d g m e n t s
We t h a n k Wei Wang, Jessica B. Wiggins, a n d t h e r e s t of t h e Chakraborty M, Baldwin-Brown JG, Long AD, Emerson JJ. Contiguous a n d accurate de novo assembly of metazoan genomes with modest long read coverage. Nucleic Acids Res. 2016;44(19):e147. Chakraborty M, Emerson J, Nirale N. Drosophila sechellia (sech25) [Direct Submission]. Departm ent of Ecology a n d Evolutionary Biology, Irvine (CA): University of California; 2017. C h a n PP, Lowe TM. tRNAscan-SE: searching for tRNA genes in genomic sequences. Methods Mol Biol. 2019;1962:1-14. Lewis-Sigler I n s t it u t e Genomics Core Facility a t Princeton Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, Markow TA, University for t h e Illumina sequencing, Dibyendu K u m a r a n d t h e r e s t of t h e W a k s m a n I nst i t u t e Genomics Core Facility a t Rutg ers University for t h e PacBio sequencing. We t h a n k Rob Kulathinal for t h e helpful discussion on t h e g e n o m e assemb ly. We a r e thankf ul to Cody Ste vens a n d Nicholas Gatt one for the i r helpful discussions on CRISPR a n d genomic DNA extraction, a n d H e a t h e r Ciallella for ass i stan c e with computi ng resources. We also acknowledge t h e Office of Advanced Research Computing (OARC) a t Rutgers, The S tate University of New Jersey for providing access to t h e Amare l cluster a n d associated resear c h computing resources t h a t h a v e contributed to t h e resu l t s rep o rted here. Funding CJS a n d NTR were partially s u p p o r t e d by t h e C enter for Computati ona l a n d Integrative Biology, Rutgers-C am den . This resear c h was s u p p o r t e d by t h e National In s t i t u t e of Ge neral K a u f m a n TC, Kellis M, Gelbart W, Iyer VN, et al. Evolution of genes a n d genomes on the Drosophila phylogeny. Nature. 2007; 450(7167):203-218. del Valle Rodriguez A, Didiano D, Desplan C. Power tools for gene expression a n d clonal analysis in Drosophila. Nat Methods. 2011; 9(1):47-55. Duffy JB. GAL4 system in Drosophila: a fly geneticist's Swiss arm y knife. Genesis. 2002;34(1-2):1-15. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time a n d space complexity. BMC Bioinformatics. 2004a; 5(113):113. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy a n d high throughpu t. Nucleic Acids Res. 2004b;32(5):1792-1797. Ferreiro MJ, Pe ´rez C, Marchesano M, Ruiz S, Caputi A, Aguilera P, Barrio R, Cantera R. Drosophila melanogaster White Mu t a n t w(1118) undergo retinal degeneration. Front Neurosci. 2017;11(732):732. Friard O, Gamba M. BORIS: a free, versatile open-source event-log-Medical Sciences of t h e National In s t it u t e s of Health ging software for video/audio coding a n d live observations. (2R15GM101597-02 to NY), a n d by t h e National Science Methods Ecol Evol. 2016;7(11):1325-1330. Foundation (IOS-1926802 to NY). Garcia C, Delprat A, Ruiz A, Valente VL. Reassignment of Drosophila willistoni genome scaffolds to chromosome II arms . G3